Summary

1. 44% of PRAME is at TSS p‹10-20 • The tumor antigen PRAME is a subunit of a Cul2 ubiquitin ligase and associates with active NFY promoters • Nawel Mahrour1*, A. Costessi2*, E. Tijchon2, R. Stunnenberg2, M.A. Stoel2, P.W. Jansen2, D. Sela1, • L. Florens1, S. Martin-Brown1, M.P. Washburn1,3, J.W. Conaway1,4, R.C. Conaway1,4 and H. Stunnenberg2 • Stowers Institute for Medical Research. Kansas City, MO 64110; 2. Dept. Molecular Biology, Radboud University, Nijmegen, The Netherlands; 3. Depts. of Pathology & Lab. Medicine and 4. Biochemistry & Mol Biology, University of Kansas Medical School, Kansas City, KS • * Equal contribution Substrate? Ub PRAME EloB E2 BC-Box Rbx1 EloC Cul2-Box Cul2 Introduction º PRAME (Preferentially expressed antigen in melanoma) is a human oncoprotein highly expressed in many tumors, but not present in normal tissues. º High PRAME expression is predictive of poor prognosis in melanoma, neuroblastoma and breast cancer. º Thus, PRAME may be a promising therapeutic target. º However, the molecular function(s) of PRAME are not yet understood. º Our aim is to elucidate PRAME’s role in oncogenesis at the molecular level. PRAME’s binding to Elongin BC depends on its BC-box PRAME’s binding to Cul2 + Rbx1 depends on its Cul2-box Multiple Sequence Alignment of PRAME with known Elongin BC- associating proteins predicts BC-box and Cul2-box motifs at PRAME’s N terminus BC-box Cul2-box Input TAG-IP BC-box mut BC-box mut Relative NSAfs WT WT TAG-PRAME Cul2 EloB PRAME pulls down all the components of a Cullin2-based ubiquitinligase RLVELAGQSL PRAME PRAME LPRELFPPL WT EloC WT LRR Tag RPVELFGQSL LRR Tag BC-box mutant AARELFPPL Cul2-box mutant BC-box Cul2-box BC-box Cul2-box PRAME complexes support poly-Ub chain formation in vitro PRAME assembles into a Cullin2-based ubiquitinligase - + + + + ATP + + + + - E1+E2 Flag-PRAME+B/C - + + + + His Cul2+Myc Rbx1 - + + + + Poly-Ub chains Flag-PRAME PRAME occupancy correlates positively with active histone marks and PolII and negatively with inactive marks Blot Flag/ Ub ChiP-Seq: PRAME is enriched at Transcription Start Sites (-87bp) PRAME is enriched at CCAAT motifs PRAME plays a role in gene activation • Summary • We applied protein-complex purification strategies (MudPIT) and showed that PRAME is the substrate recognition subunit of a Cullin2-based E3 ubiquitin ligase. • Genome-wide chromatin immunoprecipitation experiments reveal that PRAME is specifically enriched at transcriptionally active promoters that are also bound by NFY transcription factor. • Our data define a model for the biological function of PRAME, and support a role for the PRAME ubiquitin ligase complex in NFY-mediated transcriptional regulation, paving the road for understanding the functions of PRAME in normal cells and in human malignancies. PRAME colocalizes with transcription factors NFYA and NFYB